CN111778284A - 通过ctrp6介导的动脉粥样化动物模型及其建立方法 - Google Patents
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Abstract
本发明公开了一种通过CTRP6介导的动脉粥样硬化动物模型，所述动物模型通过外源注射腺病毒系统性高表达CTRP6获得。所述动物模型按照以下步骤进行：步骤1、构建腺病毒表达载体Ad‑CTRP6；步骤2、将步骤1得到的所述腺病毒载体通过尾部静脉注射到ApoE‑/‑小鼠中，注射后采用高脂饲料喂养6周即得到本发明的动物模型。本发明模型建立过程简便、成功率高、结果稳定可靠，节约了饲养实验动物的时间，还避免了模型构建失败的后果，本发明建立的动物模型在动脉粥样硬化的病因学、病理学、药物筛选、临床诊断和治疗等研究中具有重要的实际应用价值，可广泛推广。
Description
技术领域
本发明属于基础医学技术领域，具体涉及一种通过CTRP6介导的动脉 粥样化动物模型，还涉及上述动脉粥样化动物模型的建立方法。
背景技术
泡沫细胞的形成是引发动脉粥样硬化斑块形成非常重要的因素之一。当 大动脉或中动脉的某些部位内膜增厚，沉积了大量脂蛋白，单核细胞经趋化 因子MCP-1的作用，分化为巨噬细胞，巨噬细胞和少量平滑肌细胞吞噬血 液中的氧化低密度脂蛋白胆固醇形成泡沫细胞，附着于血管内皮形成斑块； 随着病程进展，斑块增大，斑块易损性增加，患者可表现为各种严重的临床 表现，如心梗等，如果斑块破裂形成血栓，患者死亡风险增加。
C1q/肿瘤坏死因子相关蛋白6(CTRP6)是一种新发现的分泌蛋白，其 结构与功能与脂联素相关。肿瘤，动脉粥样硬化和组织炎症等疾病的发病病 程都与CTRP6有关，其在各种细胞的生理活动也有着重要作用。
在对动脉粥样硬化进行实验研究时，需要制备实验动物模型。现有的技 术中，中小型动物在实验模型的构建中，一般都会使用高脂饲养以及球囊损 伤的方法，需要12周去干预实验动物，耗费的时间与人力都十分庞大。
发明内容
本发明第一个目的是提供一种通过CTRP6介导的动脉粥样硬化动物模 型，缩短了动物模型构建时间。
本发明第二个目的是提供一种通过CTRP6介导的动脉粥样硬化动物模 型的构建方法。
本发明所采用的第一种技术方案是一种通过CTRP6介导的动脉粥样硬 化动物模型，动物模型通过外源注射腺病毒系统性高表达获得。
本发明所采用的第二种技术方案是：一种通过CTRP6介导的动脉粥样 硬化动物模型的建立方法，按照以下步骤进行：
步骤1、构建腺病毒表达载体Ad-CTRP6；
步骤2、将步骤1得到的所述腺病毒载体通过尾部静脉注射到ApoE-/-小鼠中，注射后采用高脂高胆固醇饲料喂养6周，第四周时再注射一次步骤 1得到的腺病毒，6周后得到的小鼠即得到本发明的动物模型。
本发明所采用第二种技术方案的特点还在于，
步骤1中腺病毒表达载体具体构建步骤如下：
步骤1.1、采用HindⅢ、SalⅠ双酶切小鼠CTRP6 cDNA质粒，电洗脱 回收CTRP6 cDNA片段，HindⅢ、SalⅠ双酶切pCTRP6-CMV质粒，电洗 脱法回收pAdTrack-CMV HindⅢ和SalⅠ双酶切线性片段，再通过T4DNA 连接酶连接CTRP6 cDNA片段与p AdTrack-CMV HindⅢ、Sal I双酶切线性 片段，将连接得到的产物转化到E.coliDH5α感受态细胞中，经Kan+LB培养 基培养后采用碱裂解法提取重组质粒；
步骤1.2、将步骤1.1所得的p AdTrack-CTRP6重组质粒经PemⅠ酶切， 电洗脱回收线性片段，将酶切后的p AdTrack-CTRP6 PemⅠ酶切线性片段电 转法转入含p AdEasy-1的E.coli BJ5183电感受态细胞，转化产物经Kan+LB 固体培养基培养后采用碱性裂解法提取p AdEasy-CTRP6重组质粒；
步骤1.3、采用PacⅠ酶切步骤1.2所得的p AdEasy-CTRP6得到Pac I 线性化重组质粒p AdEasy-CTRP6片段，采用5μg的PacⅠ线性化重组质粒 p AdEasy-CTRP6片段转染HEK293细胞，转染后培养7～10天，每天观察 细胞生长及绿色荧光蛋白表达，当细胞中绿色荧光蛋白表达呈“彗星”状并出 现细胞病变效应时，收集细胞并涡旋震荡4次使细胞裂解，裂解后在 12000r/min的条件下离心3min，收集上清液即为Ad-CTRP6病毒液，将 Ad-CTRP6病毒液于-80℃保存备用；
步骤1.4、感染当天，将200μL所述步骤1.3得到的Ad-CTRP6病毒液 加至1.5mLDEME完全培养液中培养90min后，加入3.5mL DMEM完全 培养液培养，感染第3天，细胞出现明显细胞病变效应并伴有脱落时，收集 病毒，再重复感染Ad-CTRP6病毒液4次，将所得的病毒总和进行收集即为 本发明的腺病毒表达载体Ad-CTRP6。
步骤1.3中转染前1天将HEK293细胞于直径为60mm的培养皿培养， 转染时细胞的细胞密度30％-50％。
步骤1.4中感染前1天，将HEK293细胞于直径为100mm的培养皿， 感染时细胞的细胞密度75％-85％。
步骤2中高脂饲料的胆固醇和脂肪量为1.5％cholesterol+15％fat。
步骤2中注射入ApoE-/-小鼠的腺病毒表达载体Ad-CTRP6的浓度是 1×1011vp/mL。
本发明的有益效果是：与现有动物模型的建立方法相比，12周的实验动 物模型构建时间太长并且消耗人力物力都十分巨大，本专利所提到的动物模 型构建方法所需时间只有6周就可以构建所需的动脉粥样硬化模型。与一般 的注射外源性蛋白方法相比，腺病毒的转染方式有很明显的优势，不需要频 繁的注射蛋白只需要每3周注射一次即可，由于CTRP6基因结构序列较大 故选择腺病毒携带基因的方式最为简单方便。
附图说明
图1为本发明动物模型与对照组通过油红O染色后显微镜拍照主动脉 弓斑块时的对比示意图；
图2为本发明通过计算各组斑块面积后所得到的统计对比图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本发明是一种通过CTRP6介导的动脉粥样硬化动物模型，所述动物模 型可通过外源注射腺病毒系统性高表达CTRP6的得到，本发明制备的动物 模型的造模时间相比于现有的造模时间明显缩短。
本发明是一种通过CTRP6介导的动脉粥样化动物模型的构建方法，具 体包括以下步骤：
步骤1、制备Ad-CTRP6基因的腺病毒表达载体
步骤1.1、采用HindⅢ、SalⅠ双酶切小鼠CTRP6 cDNA质粒，1％琼脂 糖凝胶平板电泳法检测双酶切产物，电洗脱回收CTRP6 cDNA片段，采用 HindⅢ和SalⅠ双酶切pCTRP6-CMV质粒，电洗脱法回收pAdTrack-CMV HindⅢ、SalⅠ双酶切线性片段，再通过T4DNA连接酶连接CTRP6 cDNA 片段与pAdTrack-CMV HindⅢ、SalⅠ双酶切线性片段，将连接得到的产物 转化到E.coliDH5α感受态细胞中，经Kan+LB培养基培养后采用碱裂解法提 取得到pAdTrack-CTRP6重组质粒，通过HindⅢ、SalⅠ双酶切鉴定上述重 组质粒。
步骤1.2、将步骤1.1所得的p AdTrack-CTRP6重组质粒经PemⅠ酶切， 电洗脱回收线性片段，将酶切后的p AdTrack-CTRP6PemⅠ酶切线性片段电 转法转入含p AdEasy-1的E.coli BJ5183电感受态细胞，转化产物经Kan+LB 固体培养基培养后采用碱性裂解法提取pAdEasy-CTRP6重组质粒，分别通 过BamHⅠ和PacⅠ酶切鉴定pAdEasy-CTRP6重组质粒，并对pAdEasy-CTRP6重组质粒行目的基因序列测定(Invitrogen)。
步骤1.3、采用PacⅠ酶切步骤1.2所得的pAdEasy-CTRP6得到Pac I线 性化重组质粒p AdEasy-CTRP6片段，转染前1天将HEK293细胞于直径为 60mm的培养皿培养，采用5μg的Pac I线性化重组质粒pAdEasy-CTRP6片 段转染HEK293细胞，转染时细胞密度30％-50％，转染后培养细胞7～10 天，每天观察细胞生长及细胞中绿色荧光蛋白(GFP)表达情况，当GFP表 达呈“彗星”状并出现细胞病变效应(CPE)时，收集细胞于15mL离心管中， 采用涡旋震荡细胞4次使细胞裂解，裂解后在12000r/min的条件下离心3min， 将离心后的上清液分装至1.5mL离心管中，所述上清液即为Ad-CTRP6病毒 液，将Ad-CTRP6病毒液于-80℃保存备用。
步骤1.4、感染前1天，将HEK293细胞于直径为100mm的培养皿培养， 感染当天，将200μL所述步骤1.3得到的Ad-CTRP6病毒液加至1.5mL DEME 完全培养液中培养90min后，加入3.5mL DMEM完全培养液培养，感染第 3天，细胞出现明显细胞病变效应并伴有脱落时，收集病毒，再重复感染 Ad-CTRP6病毒液4次，将所得的病毒总和进行收集即为本发明的腺病毒表 达载体Ad-CTRP6。
步骤2、ApoE-/-小鼠尾静脉注射步骤1.5的腺病毒表达载体Ad-CTRP6 后，将ApoE-/-小鼠采用高脂饲料喂养6周，第四周时再注射一次步骤1.5的 腺病毒表达载体Ad-CTRP6，得到的小鼠即为本发明的动物模型。高脂饲料 的胆固醇和脂肪量均为1.5％cholesterol+15％fat。
上述ApoE-/-小鼠尾部静脉注射时腺病毒表达载体Ad-CTRP6的浓度是 1×1011vp/mL。
模型验证：
1.实验对象
(1)选用本发明制备好的动物模型为试验组(Ad-CTRP6)。
(2)ApoE-/-小鼠尾部静脉注射200μL生理盐水后采用高脂饲料 喂养6周及得到空白对照组(saline)。
(3)ApoE-/-小鼠尾静脉注射Ad-GFP腺病毒，腺病毒的病毒数量为 1×1011vp/mL，将注射后的小鼠采用高脂饲料喂养6周，在第四周时再注射 一次Ad-GFP，得到的小鼠即为对照组(Ad-GFP)，高脂饲料的胆固醇和脂 肪量均为1.5％cholesterol+15％fat。
上述Ad-GFP腺病毒具体按照以下步骤制备：
步骤a、将pRNAT-H1.1/Adeno用PmeI进行酶切，然后通过低熔点琼脂 糖凝胶电脉回收后，再用碱性磷酸酶CIAP去磷酸化、苯酚/氯仿抽提后，转 化入含有腺病毒骨架载体pAdeasy-1的超感受态BJ5183中，通过细菌内同 源重组法获得的阳性重组质粒命名为pAd-GFP。
步骤b、将经PacI线性化的阳性重组质粒pAd-GFP进行苯酚/氯 仿抽提，乙醇/醋酸钠共沉淀后加入40μL消毒三蒸水溶解，待完全溶 解后加入等体积的阳离子脂质体Lipofectamine2000，于37℃孵育4h 后，由阳离子脂质体Lipofectamine2000介导转染HEK293细胞细胞， 置37℃、5％CO2的培养箱中孵育14～20天后即可观察到病毒蚀斑的 出现。
步骤c、将步骤b得到的重组腺病毒在12000r/min的转速下离心 3次后，取离心后的重组腺病毒上清液500μL，加入90％融合的 HEK293细胞中，于37℃、5％CO2培养箱中培养，显微镜下观察到 95％～100％细胞出现细胞病变后，离心收集细胞，并将细胞于-80℃和37℃之间反复冻融3次，以3000rpm/min离心5min收集上清。对 重组腺病毒上清进行CsCl密度梯度离心纯化两次，并将纯化后的腺
2.验证步骤：
饲喂6周后，对空白对照组、对照组和实验组均腹腔注射1％戊巴比妥钠 安乐处死，分离其各组主动脉冷冻保存。将冷冻保存的三组主动脉置于室温 解冻30min，然后放于4％的多聚甲醛中固定15min，在置于双蒸水中两次(一 次一分钟)，取出后放在60％的异丙醇中5min，之后放于油红O染液中30min， 染色结束依次放入60％的异丙醇与双蒸水中各1min，之后用水溶性封片剂 封片，并且置于显微镜下计算斑块面积，图1左侧为对照组(saline)的小 鼠动物模型剥离的主动脉，图1中间为对照组(Ad-GFP)小鼠动物模型剥 离的主动脉，图1右侧为实验组(Ad-CTRP6)小鼠动物模型剥离的主动脉， 由图1对比可知，从实验组(Ad-CTRP6)小鼠动物模型剥离的主动脉上斑 块面积大于空白对照组(saline)和对照组(Ad-GFP)小鼠动物模型剥离的 主动脉上斑块面积，由图2可知：横坐标表示空白对照组(saline)，对照组 (Ad-GFP)和实验组(Ad-CTRP6)的编号；纵坐标表示小鼠总损伤面积的大小， 单位长度为10mm2，计量单位为平方毫米；可以看到两组对照组的总损伤面 积约为14mm2，其损伤面积小于实验组所构建的动物模型所产生的总损伤面 积约为31mm2。说明本发明通过CTRP6加速动脉粥样硬化形成的动物模型 制备成功，且表型明显。
由于与正常的动物模型的建立相比本实验需要使用腺病毒进行干预，为 了确定不是腺病毒导致实验动物出现明显的动脉粥样硬化过程，故使用GFP 荧光蛋白与Ad-CTRP6做对比，可以排除腺病毒对实验动物的影响，此外还 可以通过观察荧光反应确定腺病毒成功感染了实验动物。
本发明是一种体外注射腺病毒携带CTRP6蛋白基因通过循环系统去 快速制备动脉粥样硬化实验动物模型的方法。通过观察小鼠主动脉上斑块 的形成量，从而去检测CTRP6在动脉粥样硬化斑块实验动物模型构建中的 作用，利用CTRP6的功能快速制备动脉粥样硬化的实验动物模型。本发明 模型建立过程简便、成功率高、结果稳定可靠，节约了饲养实验动物的时 间，还避免了模型构建失败的后果，本发明建立的动物模型在动脉粥样硬化的病因学、病理学、药物筛选、临床诊断和治疗等研究中具有重要的实 际应用价值，可广泛推广。
Claims (7)
1.一种通过CTRP6介导的动脉粥样硬化动物模型，其特征在于，所述动物模型通过腺病毒系统性高表达可诱导小鼠CTRP6的表达得到。
2.一种权利要求1所述的一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，其特征在于，按照以下步骤进行：
步骤1、构建腺病毒表达载体Ad-CTRP6；
步骤2、将步骤1得到的所述腺病毒载体Ad-CTRP6通过尾部静脉注射到ApoE-/-小鼠中，注射后采用高脂饲料喂养6周，第四周时再注射一次腺病毒表达载体Ad-CTRP6，6周后得到的小鼠即得到本发明的动物模型。
3.根据权利要求2所述的一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，其特征在于，所述步骤1中腺病毒表达载体具体构建步骤如下：
步骤1.1、采用Hind Ⅲ、Sal Ⅰ双酶切小鼠CTRP6 cDNA质粒，电洗脱回收CTRP6 cDNA片段，Hind Ⅲ、Sal Ⅰ双酶切pCMV-CTRP6质粒，电洗脱法回收pAdTrack-CMV Hind Ⅲ和Sal Ⅰ双酶切线性片段，再通过T4DNA连接酶连接CTRP6 cDNA片段与pAdTrack-CMV Hind Ⅲ、SalI双酶切线性片段，将连接得到的产物转化到E.coliDH5α感受态细胞中，经Kan+LB培养基培养后采用碱裂解法提取重组质粒；
步骤1.2、将步骤1.1所得的pAdTrack-CTRP6重组质粒经Pem Ⅰ酶切，电洗脱回收线性片段，将酶切后的p AdTrack-CTRP6 Pem Ⅰ酶切线性片段电转法转入含pAdEasy-1的E.coliBJ5183电感受态细胞，转化产物经Kan+LB固体培养基培养后采用碱性裂解法提取pAdEasy-CTRP6重组质粒；
步骤1.3、采用PacⅠ酶切步骤1.2所得的pAdEasy-CTRP6得到Pac I线性化重组质粒pAdEasy-CTRP6片段，采用5μg的Pac Ⅰ线性化重组质粒p AdEasy-CTRP6片段转染HEK293细胞，转染后培养细胞7～10天，每天观察细胞生长及绿色荧光蛋白表达，当细胞中绿色荧光蛋白表达呈“彗星”状并出现细胞病变效应时，收集HEK293细胞并涡旋震荡4次使细胞裂解，裂解后在12000r/min的条件下离心3min，收集上清液即为Ad-CTRP6病毒液，将Ad-CTRP6病毒液于-80℃保存备用；
步骤1.4、感染当天，将200μL所述步骤1.3得到的Ad-CTRP6病毒液加至1.5mL DEME完全培养液中培养90min后，加入3.5mL DMEM完全培养液培养，感染第3天，细胞出现明显细胞病变效应并伴有脱落时，收集病毒，再重复感染Ad-CTRP6病毒液4次，将所得的病毒总和进行收集即为本发明的腺病毒表达载体Ad-CTRP6。
4.根据权利要求3所述的一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，其特征在于，所述步骤1.3中转染前1天将HEK293细胞于直径为60mm的培养皿培养，转染时细胞的细胞密度30％-50％。
5.根据权利要求3所述的一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，其特征在于，所述步骤1.4中感染前1天，将HEK293细胞于直径为100mm的培养皿，感染时细胞密度75％-85％。
6.根据权利要求2所述的一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，其特征在于，所述步骤2中高脂饲料的胆固醇和脂肪量为1.5％cholesterol+15％fat。
7.根据权利要求2所述的一种通过CTRP6介导的动脉粥样化动物模型的建立方法，其特征在于，所述步骤2中注射入ApoE-/-小鼠的腺病毒表达载体Ad-CTRP6的浓度是1×1011vp/mL。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010039666A1 (en) * | 1999-01-11 | 2001-11-08 | David A. Dichek | Non-human mammalian model for atherosclerosis and methods for screening agents for use in the treatment of atherosclerosis |
| US20080160034A1 (en) * | 2004-08-25 | 2008-07-03 | Genentech, Inc. | Novel Gene Disruptions, Compositions and Methods Relating Thereto |
| EP2708233A1 (en) * | 2011-05-13 | 2014-03-19 | The University of Tokyo | Ctrp6 which can be used as therapeutic and prophylactic agent for autoimmune diseases |
| US20180230489A1 (en) * | 2015-10-28 | 2018-08-16 | Voyager Therapeutics, Inc. | Regulatable expression using adeno-associated virus (aav) |
-
2020
- 2020-06-24 CN CN202010584843.6A patent/CN111778284B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010039666A1 (en) * | 1999-01-11 | 2001-11-08 | David A. Dichek | Non-human mammalian model for atherosclerosis and methods for screening agents for use in the treatment of atherosclerosis |
| US20080160034A1 (en) * | 2004-08-25 | 2008-07-03 | Genentech, Inc. | Novel Gene Disruptions, Compositions and Methods Relating Thereto |
| EP2708233A1 (en) * | 2011-05-13 | 2014-03-19 | The University of Tokyo | Ctrp6 which can be used as therapeutic and prophylactic agent for autoimmune diseases |
| US20140194343A1 (en) * | 2011-05-13 | 2014-07-10 | The University Of Tokyo | Ctrp6 which can be used as therapeutic and prophylactic agent for autoimmune diseases |
| US20180230489A1 (en) * | 2015-10-28 | 2018-08-16 | Voyager Therapeutics, Inc. | Regulatable expression using adeno-associated virus (aav) |
Non-Patent Citations (5)
| Title |
|---|
| XIAOZHEN ZHUO ET AL.: "Flavone of Hippophae (H-flavone) lowers atherosclerotic risk factors via upregulation of the adipokine C1q/tumor necrosis factor-related protein 6 (CTRP6) in macrophages" * |
| 李嵘;杨晓霞;于艳;周美兰;田秀娟;冯世栋;王汉民;: "C1q/肿瘤坏死因子相关蛋白6(CTRP6)介导庆大霉素所致大鼠急性肾损伤" * |
| 毋文静 * |
| 王雪梅;魏琴;张春;姜涛;段明军;杨毅宁;: "重组腺病毒介导脂联素基因对ApoE~(-/-)小鼠动脉粥样硬化的作用机制" * |
| 陈闽;何姜;刘小莺;黄敬泽;王林曦;刘礼斌;: "携带人脂联素基因重组腺病毒的构建及表达" * |
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| CN111778284B (zh) | 2023-09-22 |
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